Compositions and methods for detection of beta n methylamino l alanine in cu zn superoxide dismutase 1

ABSTRACT

Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify β-N-methylamino-L-alanine (BMAA) in the Cu/Zn Superoxide Dismutase 1 (SOD1) protein. Further provided are methods for detecting, preventing, or treating amyotrophic lateral sclerosis in a subject using isotopically labeled reagents to detect and/or quantify β-N-methylamino-L-alanine (BMAA) in the Cu/Zn Superoxide Dismutase 1 (50D1) protein. Further provided are isotopically labeled reagents and methods for detecting BMAA in additional proteins from patient samples.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/368,437, filed Jul. 29, 2016, and U.S.Provisional Patent Application Ser. No. 62/368,562, filed Jul. 29, 2016,each of which are expressly incorporated herein by reference.

BACKGROUND

The non-protein amino acid β-N-Methylamino-L-Alanine (BMAA) isbiosynthetically produced in cyanobacteria. Human exposure to thisunnatural amino acid has been linked to neurological disorders,including amyotrophic lateral sclerosis (ALS) and parkinsonism dementiacomplex (PDC) like symptoms. Dietary exposure of BMAA in primates hasshown neurofibrillary tangles (NFT) and β-amyloid plaques, hallmarksigns of neuropathological disease. Additionally, cell culture studieshave shown that exogenous exposure to BMAA can result in incorporationof this non-protein amino acid in place of L-serine. Supplementaltreatment with L-serine has been shown to reduce the rate of BMAAincorporation and regression of neuropathological symptoms.

While these findings suggest that BMAA can be incorporated intoproteins, efforts to further study the role of BMAA in biologicalsystems has been hampered by the limited availability of suitableanalytical probes and methods. Improved analytical tools and methods areneeded to fully understand the qualitative and quantitative nature ofincorporation of BMAA in proteins, particularly human proteins, as wellas the mechanism and functional consequences of this process.

Because of the lack of appropriate reagents and methods, theidentification of endogenous BMAA incorporation in patients sufferingfrom neurological disorders, including amyotrophic lateral sclerosis(ALS), has not yet been shown. Thus, improved compositions and methodsare needed for the detection of BMAA incorporation into proteinsimplicated in neurological disorders.

SUMMARY

Provided herein are isotopically labeled reagents, includingisotopically labeled small molecules and peptides, that can be used todetect and/or quantify β-N-methylamino-L-alanine (BMAA) in the Cu/ZnSuperoxide Dismutase 1 (SOD1) protein. Using these novel reagents, theinventors have identified the incorporation of BMAA into the SOD1protein in samples from patients suffering from amyotrophic lateralsclerosis (ALS). The reagents can be used as stable isotope labeledstandards in analytical methods, including in conjunction with massspectrometry, to detect and/or quantify BMAA in a sample, such as aprotein sample from a subject. In addition, the inventors have developedisotopically labeled reagents that can detect the incorporation of BMAAinto several additional proteins in patient samples.

In one aspect, provided herein is a polypeptide comprising a sequencethat is at least 85% identical to SEQ ID NO: 1, wherein amino acidposition 107 comprises a BMAA residue which is isotopically labeled andis defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least 100.

In one aspect, provided herein is a polypeptide comprising a sequencethat is at least 85% identical to SEQ ID NO: 2, wherein amino acidposition 107 comprises a BMAA residue which is isotopically labeled andis defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least 100.

In another aspect, provided herein is a method for detectingβ-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising (a)purifying SOD1 protein from a biological specimen to provide a purifiedSOD1 protein sample, (b) spiking the purified SOD1 protein sample with adefined amount of an isotopically labeled compound defined by theformula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and(d) measuring BMAA levels in the SOD1 protein sample by isotope dilutionanalysis.

In one aspect, provided herein is a method for detectingβ-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising: (a)purifying SOD1 protein from a biological specimen to provide a purifiedSOD1 protein sample; (b) spiking the purified SOD1 protein sample with adefined amount of a polypeptide, wherein the polypeptide includes one ormore isotopically labeled β-N-methylamino-L-alanine (BMAA) residues,wherein each of the one or more isotopically labeled BMAA residues isisotopically labeled with one or more stable isotopes; to provide aBMAA-spiked sample; (c) analyzing the BMAA-spiked sample by massspectrometry; and (d) measuring BMAA levels in the SOD1 protein sampleby isotope dilution analysis.

In a further aspect, provided herein is a method of detecting orpredicting amyotrophic lateral sclerosis in a subject, comprising (a)purifying SOD1 protein from a biological specimen from a subject toprovide a purified SOD1 protein sample; (b) spiking the purified SOD1protein sample with a defined amount of an isotopically labeled compounddefined by the formula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d)measuring BMAA level in the SOD1 protein sample by isotope dilutionanalysis; and (e) identifying the subject for the presence or risk ofamyotrophic lateral sclerosis if the BMAA level in the SOD1 protein isgreater than a normal reference value.

In another aspect, provided herein is a method of detecting orpredicting amyotrophic lateral sclerosis in a subject, comprising (a)purifying SOD1 protein from a biological specimen from a subject toprovide a purified SOD1 protein sample; (b) spiking the purified SOD1protein sample with a defined amount of a polypeptide, wherein thepolypeptide includes one or more isotopically labeledβ-N-methylamino-L-alanine (BMAA) residues, wherein each of the one ormore isotopically labeled BMAA residues is isotopically labeled with oneor more stable isotopes, to provide a BMAA-spiked sample; (c) analyzingthe BMAA-spiked sample by mass spectrometry; (d) measuring BMAA level inthe SOD1 protein sample by isotope dilution analysis; and (e)identifying the subject for the presence or risk of amyotrophic lateralsclerosis if the BMAA level in the SOD1 protein is greater than a normalreference value.

In one aspect, provided herein is a method of preventing or treatingamyotrophic lateral sclerosis in a subject, comprising (a) purifyingSOD1 protein from a biological specimen from a subject to provide apurified SOD1 protein sample; (b) spiking the purified SOD1 proteinsample with a defined amount of an isotopically labeled compound definedby the formula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d)measuring BMAA levels in the SOD1 protein sample by isotope dilutionanalysis; (e) identifying the subject for the presence or risk ofamyotrophic lateral sclerosis if the BMAA levels in the SOD1 protein isgreater than a normal reference value; and (f) administering to thesubject L-serine in an amount sufficient to prevent or treat theamyotrophic lateral sclerosis.

In one final aspect, provided herein is a method of preventing ortreating amyotrophic lateral sclerosis in a subject, comprising (a)purifying SOD1 protein from a biological specimen from a subject toprovide a purified SOD1 protein sample; (b) spiking the purified SOD1protein sample with a defined amount of a polypeptide, wherein thepolypeptide includes one or more isotopically labeledβ-N-methylamino-L-alanine (BMAA) residues, wherein each of the one ormore isotopically labeled BMAA residues is isotopically labeled with oneor more stable isotopes, to provide a BMAA-spiked sample; (c) analyzingthe BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levelsin the SOD1 protein sample by isotope dilution analysis; (e) identifyingthe subject for the presence or risk of amyotrophic lateral sclerosis ifthe BMAA levels in the SOD1 protein is greater than a normal referencevalue; and (f) administering to the subject L-serine in an amountsufficient to prevent or treat the amyotrophic lateral sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of an example procedure used to convert aphosphoserine-containing peptide to BMAA-containing peptide. The peptidesequence shown in FIG. 1 is unique to superoxide dismutase 1 (SOD1).

FIGS. 2A-2B illustrate the intact mass of stable isotope-labeled (SIL)phosphopeptide substrate and BMAA peptide product.

FIG. 3 illustrates the tandem mass spectrum of the phosphorylatedserine-containing stable isotope-labeled peptide.

FIG. 4 illustrates the tandem mass spectrum of a BMAA-containing stableisotope-labeled peptide, confirming localization of modification.

FIG. 5 demonstrates that co-eluting fragments confirm identity of theBMAA-containing peptide. BMAA- and phosphoserine-containing peptideshave distinct retention times. The BMAA-containing peptide appears tohave two peaks which suggests L and D isomers of the BMAA peptide havebeen produced.

FIG. 6 illustrates the conserved peak area of fragments specific to SILBMAA peptide that allow for confident identification of endogenouspeptide.

FIG. 7 is a plot showing that the elution time of SIL BMAA peptide isnecessary for correct identification of endogenous peptide. Withoutthis, the incorrect peak which is also within 5 ppm mass accuracy of theBMAA peptide of interest might be selected.

FIG. 8 demonstrates the ability of mass spectrometry to accuratelyidentify endogenous BMAA-containing peptide in SOD1 protein digestobtained from ALS erythrocytes.

FIG. 9 is a plot illustrating the co-elution of endogenous and stableisotope-labeled BMAA containing peptides. Co-elution aids in confidentidentification.

FIG. 10 is a schematic showing the method of filter aided samplepreparation for bottom-up protein characterization.

FIG. 11 is a schematic showing the post-translational modificationsidentified with high confidence in SOD1. The amino acid numbering shownin the figure does not include the starting methionine.

FIG. 12 is a schematic showing the post-translational modificationsidentified with medium confidence in SOD1. The amino acid numberingshown in the figure does not include the starting methionine.

FIG. 13 illustrates the tandem mass spectrum of a BMAA-containing stableisotope-labeled peptide. Fragment ions corresponding to the endogenouspeptide with BMAA were found at the expected location, confirminglocalization of modification.

FIG. 14 illustrates the tandem mass spectrum of BMAA containing stableisotope-label (¹³C₄ ¹⁵N₂ β-N-Methylamino-L-Alanine, or Compound 3).

DETAILED DESCRIPTION

Provided herein are isotopically labeled reagents, includingisotopically labeled small molecules and peptides, that can be used todetect and/or quantify β-N-methylamino-L-alanine (BMAA) in the Cu/ZnSuperoxide Dismutase 1 (SOD1) protein. Using these novel reagents, theinventors have identified the incorporation of endogenous BMAA into theSOD1 protein in samples from patients suffering from amyotrophic lateralsclerosis (ALS). The reagents can be used as stable isotope labeledstandards in analytical methods, including in conjunction with massspectrometry, to detect and/or quantify BMAA in a sample, such as aprotein sample from a subject. In addition, the inventors have developedisotopically labeled reagents that can detect the incorporation of BMAAinto several additional proteins in patient samples.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.Amino acids include Alanine (Ala, A), Asparagine (Asn, N), Cysteine(Cys, C), Glutamine (Gln, Q), Glycine (Gly, G), Isoleucine (Ile, I),Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Proline(Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W),Tyrosine (Tyr, Y), Valine (Val, V), Histidine (His, H), Arginine (Arg,R), Lysine (Lys, K), Aspartic acid (Asp, D), and Glutamic acid (Glu, E).β-N-methylamino-L-alanine (BMAA) is represented using the letter B in apeptide sequence, unless otherwise indicated in the text.

As used herein, a “normal reference value” refers to a numerical valuethat is determined experimentally from an unaffected individual, or apopulation of unaffected individuals, for comparison to the value froman affected individual, or population of affected individuals (forexample, individuals affected by ALS). In some embodiments, the “normalreference value” being measured is from a healthy control subject thatis not affected by ALS, or from a population of healthy control subjectsthat are not affected by ALS.

Compounds and Compositions

Provided herein is a polypeptide comprising a sequence that is at least85% identical to SEQ ID NO: 1, wherein amino acid position 107 comprisesa BMAA residue which is isotopically labeled and is defined by theformula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least 100.

In one embodiment, the ¹³C isotopic enrichment factor for ^(d)C is atleast 80 and the ¹⁵N isotopic enrichment factor for N is at least 200.In one embodiment, the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C,and ^(c)C is at least 25. In one embodiment, ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, and ^(c)C is at least 80. In one embodiment,¹⁵N isotopic enrichment factor for ^(e)N is at least 100. In oneembodiment, ¹⁵N isotopic enrichment factor for ^(e)N is at least 200.

In one embodiment, the sequence is at least 90% identical to SEQ IDNO: 1. In one embodiment, the sequence is at least 95% identical to SEQID NO: 1. In one embodiment, the sequence is at least 99% identical toSEQ ID NO: 1. In one embodiment, the sequence is identical to SEQ ID NO:1.

In one aspect, provided herein is a polypeptide comprising a sequencethat is at least 85% identical to SEQ ID NO: 2, wherein amino acidposition 107 comprises a BMAA residue which is isotopically labeled andis defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least 100.

In one embodiment, the ¹³C isotopic enrichment factor for ^(d)C is atleast 80 and the ¹⁵N isotopic enrichment factor for N is at least 200.In one embodiment, the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C,and ^(c)C is at least 25. In one embodiment, the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, and ^(c)C is at least 80. In one embodiment,the ¹⁵N isotopic enrichment factor for ^(e)N is at least 100. In oneembodiment, the ¹⁵N isotopic enrichment factor for ^(e)N is at least200.

In one embodiment, the sequence is at least 90% identical to SEQ ID NO:2. In one embodiment, the sequence is at least 95% identical to SEQ IDNO: 2. In one embodiment, the sequence is at least 99% identical to SEQID NO: 2. In one embodiment, the sequence is identical to SEQ ID NO: 2.

In one embodiment, the sequence is at least 90% identical to SEQ ID NO:3. In one embodiment, the sequence is at least 95% identical to SEQ IDNO: 3. In one embodiment, the sequence is at least 99% identical to SEQID NO: 3. In one embodiment, the sequence is identical to SEQ ID NO: 3.

SEQ ID NO: 1 = Human SOD1 protein sequence with BMAA at position 107ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLBGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIG IAQNote that the BMAA sequence at position 107 (in bold) replaces theserine that is normally present in the wild type SOD1 sequence in SEQ IDNO: 1. The BMAA occurs at position 107 when the initial methionine isnot included in the peptide sequence, but occurs at position 108 whenthe initial methionine is included in the sequence.

Additional sequences for polypeptides that can be used as reagents inmethods for detection of BMAA incorporation into the SOD1 proteininclude:

SEQ ID NO: 2 = DGVADVSIEDSVISLBGDH C IIGR SEQ ID NO: 3 =HVGDLGNVTADKDGVADVSIEDSVISLBGDH C IIGR (1 missed cleavage) Where  C is Carbamidomethylated Cysteine (alkylated during sample preparation)SEQ ID NO: 4 = Human SOD1 wild-type protein sequence.ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIG IAQ SEQ ID NO: 5 =Human SOD1 protein sequence withBMAA at position 108 (with initial methionine)MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLBGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GIAQNote that the BMAA sequence at position 108 (in bold) replaces theserine that is normally present in the wild type SOD1 sequence in SEQ IDNO: 5. The BMAA occurs at position 108 when the initial methionine isincluded in the peptide sequence, but occurs at position 107 when theinitial methionine is not included in the sequence.

In one embodiment, the sequence is at least 90% identical to SEQ ID NO:5. In one embodiment, the sequence is at least 95% identical to SEQ IDNO: 5. In one embodiment, the sequence is at least 99% identical to SEQID NO: 5. In one embodiment, the sequence is identical to SEQ ID NO: 5.

SEQ ID NO: 6 = Human SOD1 wild-type proteinsequence (with initial methionine)MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GIAQ

In one embodiment, the sequence is at least 90% identical to SEQ ID NO:6. In one embodiment, the sequence is at least 95% identical to SEQ IDNO: 6. In one embodiment, the sequence is at least 99% identical to SEQID NO: 6. In one embodiment, the sequence is identical to SEQ ID NO: 6.

In certain embodiments, at least two (e.g., at least three, at leastfour, at least five, or all six) of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,and ^(f)N are isotopically labeled with a stable isotope. In particularembodiments, all of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and ^(f)N areisotopically labeled with a stable isotope.

The term “isotopic enrichment factor,” as used herein, refers to theratio between the isotopic abundance (e.g., ¹³C, ¹⁵N, or ¹⁸O) at aspecified position in a compound and the naturally occurring abundanceof that isotope. The naturally occurring abundance of ¹³C is 1.1%. Thenaturally occurring abundance of ¹⁵N is 0.37%. The naturally occurringabundance of ¹⁸O is 0.204%.

In some embodiments, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, ^(c)C, and ^(d)C is at least 25 (27.5% ¹³C incorporation at eachposition), at least 30 (33% ¹³C incorporation at each position), atleast 35 (38.5% ¹³C incorporation at each position), at least 40 (44%¹³C incorporation at each position), at least 45 (49.5% ¹³Cincorporation at each position), at least 50 (55% ¹³C incorporation ateach position), at least 55 (60.5% ¹³C incorporation at each position),at least 60 (66% ¹³C incorporation at each position), at least 65 (71.5%¹³C incorporation at each position), at least 70 (77% ¹³C incorporationat each position), at least 75 (82.5% ¹³C incorporation at eachposition), at least 80 (88% ¹³C incorporation at each position), atleast 85 (93.5% ¹³C incorporation at each position), or at least 90 (99%¹³C incorporation at each position).

In some embodiments, the ¹⁵N isotopic enrichment factor for ^(e)N and^(f)N is at least 100 (37% ¹⁵N incorporation at each position), at least110 (40.7% ¹⁵N incorporation at each position), at least 120 (44.4% ¹⁵Nincorporation at each position), at least 130 (48.1% ¹⁵N incorporationat each position), at least 140 (51.8% ¹⁵N incorporation at eachposition), at least 150 (55.5% ¹⁵N incorporation at each position), atleast 160 (59.2% ¹⁵N incorporation at each position), at least 170(62.9% ¹⁵N incorporation at each position), at least 180 (66.6% ¹⁵Nincorporation at each position), at least 190 (70.3% ¹⁵N incorporationat each position), at least 200 (74% ¹⁵N incorporation at eachposition), at least 210 (77.7% ¹⁵N incorporation at each position), atleast 220 (81.4% ¹⁵N incorporation at each position), at least 230(85.1% ¹⁵N incorporation at each position), at least 240 (88.8% ¹⁵Nincorporation at each position), at least 250 (92.5% ¹⁵N incorporationat each position), at least 260 (96.2% ¹⁵N incorporation at eachposition), or at least 265 (98.05% ¹⁵N incorporation at each position).

In some embodiments, the polypeptides can be comprised in a composition.The composition can be, for example, a solution of the isotopicallylabeled compound in a solvent. Non-limiting examples of solvents includealiphatic solvents (e.g., pentane, hexanes, cyclohexane); aromaticand/or alkylated aromatic solvents such as benzene, toluene, xylene;hydrocarbon solvents; dichloromethane, chloroform, alcohols (e.g.,methanol, ethanol, isopropanol); esters (e.g., ethyl acetate); ketones(e.g., acetone); diethyl ether; dioxane; glycol ethers and glycol etheresters; tetrahydrofuran; dimethylformamide; acetonitrile; dimethylsulfoxide; water, saline, aqueous buffers (e.g., PBS buffer), andcombinations thereof. In certain examples, the composition can comprisean aqueous solution of the compound.

In some embodiments, the isotopically labeled compound can comprise atleast 0.5% by weight (e.g., at least 1% by weight, at least 1.5% byweight, at least 2% by weight, at least 2.5% by weight, at least 3% byweight, at least 3.5% by weight, at least 4% by weight, at least 4.5% byweight, or at least 1% by weight of the composition.

The isotopically labeled compounds described above can be prepared usingmethods known in the art. Representative methodologies for thepreparation of certain active agents are described below. Theappropriate route for synthesis of a given compound agent can beselected in view of the structure of the compound as a whole as itrelates to compatibility of functional groups, protecting groupstrategies, and the presence of labile bonds. In addition to thesynthetic methodologies discussed below, alternative reactions andstrategies useful for the preparation of the compounds disclosed hereinare known in the art. See, for example, March, “Advanced OrganicChemistry,” 5^(th) Edition, 2001, Wiley-Interscience Publication, NewYork).

Isotopically labeled amino acids, such as ¹³C/¹⁵N-labeled asparagine,are commercially available, and can serve as convenient startingmaterials for the isotopically labeled compounds described herein.Scheme 1 below illustrates an example method for the preparation of BMAAfrom asparagine. Compounds having a desired isotopic labeling (e.g.,incorporating stable isotopes at particular positions within thecompound) can be prepared by selecting reagents that include stableisotope labels at the appropriate positions with their framework (e.g.,¹³C/¹⁵N-labeled asparagine).

In some embodiments, the BMAA amino acid residue is isotopicallylabeled. In some embodiments, the BMAA amino acid residue is unlabeledand the peptide is isotopically labeled at an amino acid other than atthe BMAA residue.

In some embodiments, provided herein is a polypeptide comprising asequence that is at least 85% identical to SEQ ID NO: 1, wherein aminoacid position 107 comprises a BMAA residue, wherein the polypeptide isisotopically labeled. In some embodiments, provided herein is apolypeptide comprising a sequence that is at least 85% identical to SEQID NO: 1, wherein amino acid position 107 comprises a BMAA residue,wherein the polypeptide is isotopically labeled and the isotopic labelis enriched in comparison to the corresponding naturally occurringpolypeptide.

Also provided are compositions that include one or more isotopicallylabeled SOD1 polypeptides of SOD1 peptide fragments. For example,compositions comprising a SOD1 polypeptide that includes one or moreisotopically labeled β-N-methylamino-L-alanine (BMAA) residues areprovided herein. The isotopically labeled SOD1 polypeptide can compriseat least 0.5% by weight of the composition. Each of the one or moreisotopically labeled BMAA residues can be isotopically labeled with oneor more (e.g., two or more) stable isotopes. For example, each of theone or more isotopically labeled BMAA residues can be defined by theformula below

where the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andthe ¹⁵N isotopic enrichment factor for ^(f)N is at least 100.

In some embodiments, the ¹³C isotopic enrichment factor for ^(d)C is atleast 25 (27.5% ¹³C incorporation at each position), at least 30 (33%¹³C incorporation at each position), at least 35 (38.5% ¹³Cincorporation at each position), at least 40 (44% ¹³C incorporation ateach position), at least 45 (49.5% ¹³C incorporation at each position),at least 50 (55% ¹³C incorporation at each position), at least 55 (60.5%¹³C incorporation at each position), at least 60 (66% ¹³C incorporationat each position), at least 65 (71.5% ¹³C incorporation at eachposition), at least 70 (77% ¹³C incorporation at each position), atleast 75 (82.5% ¹³C incorporation at each position), at least 80 (88%¹³C incorporation at each position), at least 85 (93.5% ¹³Cincorporation at each position), or at least 90 (99% ¹³C incorporationat each position).

In some embodiments, the ¹⁵N isotopic enrichment factor for ^(f)N is atleast 100 (37% ¹⁵N incorporation at each position), at least 110 (40.7%¹⁵N incorporation at each position), at least 120 (44.4% ¹⁵Nincorporation at each position), at least 130 (48.1% ¹⁵N incorporationat each position), at least 140 (51.8% ¹⁵N incorporation at eachposition), at least 150 (55.5% ¹⁵N incorporation at each position), atleast 160 (59.2% ¹⁵N incorporation at each position), at least 170(62.9% ¹⁵N incorporation at each position), at least 180 (66.6% ¹⁵Nincorporation at each position), at least 190 (70.3% ¹⁵N incorporationat each position), at least 200 (74% ¹⁵N incorporation at eachposition), at least 210 (77.7% ¹⁵N incorporation at each position), atleast 220 (81.4% ¹⁵N incorporation at each position), at least 230(85.1% ¹⁵N incorporation at each position), at least 240 (88.8% ¹⁵Nincorporation at each position), at least 250 (92.5% ¹⁵N incorporationat each position), at least 260 (96.2% ¹⁵N incorporation at eachposition), or at least 265 (98.05% ¹⁵N incorporation at each position).

Optionally, ^(a)C, ^(b)C, and/or ^(c)C can also be labeled with a stableisotope. In some cases, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, and ^(c)C can be at least 25 (27.5% ¹³C incorporation at eachposition), at least 30 (33% ¹³C incorporation at each position), atleast 35 (38.5% ¹³C incorporation at each position), at least 40 (44%¹³C incorporation at each position), at least 45 (49.5% ¹³Cincorporation at each position), at least 50 (55% ¹³C incorporation ateach position), at least 55 (60.5% ¹³C incorporation at each position),at least 60 (66% ¹³C incorporation at each position), at least 65 (71.5%¹³C incorporation at each position), at least 70 (77% ¹³C incorporationat each position), at least 75 (82.5% ¹³C incorporation at eachposition), at least 80 (88% ¹³C incorporation at each position), atleast 85 (93.5% ¹³C incorporation at each position), or at least 90 (99%¹³C incorporation at each position).

Optionally, ^(e)N can also be labeled with a stable isotope. In somecases, the ¹⁵N isotopic enrichment factor for ^(e)N is at least 100 (37%¹⁵N incorporation at each position), at least 110 (40.7% ¹⁵Nincorporation at each position), at least 120 (44.4% ¹⁵N incorporationat each position), at least 130 (48.1% ¹⁵N incorporation at eachposition), at least 140 (51.8% ¹⁵N incorporation at each position), atleast 150 (55.5% ¹⁵N incorporation at each position), at least 160(59.2% ¹⁵N incorporation at each position), at least 170 (62.9% ¹⁵Nincorporation at each position), at least 180 (66.6% ¹⁵N incorporationat each position), at least 190 (70.3% ¹⁵N incorporation at eachposition), at least 200 (74% ¹⁵N incorporation at each position), atleast 210 (77.7% ¹⁵N incorporation at each position), at least 220(81.4% ¹⁵N incorporation at each position), at least 230 (85.1% ¹⁵Nincorporation at each position), at least 240 (88.8% ¹⁵N incorporationat each position), at least 250 (92.5% ¹⁵N incorporation at eachposition), at least 260 (96.2% ¹⁵N incorporation at each position), orat least 265 (98.05% ¹⁵N incorporation at each position).

In some embodiments, the polypeptide can include a single isotopicallylabeled BMAA residue. In other embodiments, the polypeptide can includetwo or more isotopically labeled BMAA residues (e.g., three or moreisotopically labeled BMAA residues, four or more isotopically labeledBMAA residues, five or more isotopically labeled BMAA residues, or tenor more isotopically labeled BMAA residues). In some embodiments, theBMAA residue is substituted for Ser107 (numbering does not include thestarting methionine.

In some embodiments, the polypeptide can be defined by the formula below

where m is an integer from 0 to 8384 and n is an integer from 0 to 8384,with the proviso that at least one of m and n is not 0; the ¹³C isotopicenrichment factor for ^(d)C is at least 25; the ¹⁵N isotopic enrichmentfactor for N is at least 100; and independently for each occurrence inthe polypeptide, R₁ is H and R₂ is selected from one of the following

or R₁ and R₂, together with the atoms to which they are attached, form afive-membered heterocycle defined by the structure below

In some embodiments, the polypeptide can be defined by the formula below

where m is an integer from 0 to 108 and n is an integer from 0 to 46,with the proviso that at least one of m and n is not 0; the ¹³C isotopicenrichment factor for ^(d)C is at least 25; the ¹⁵N isotopic enrichmentfactor for N is at least 100; and independently for each occurrence inthe polypeptide, R₁ is H and R₂ is selected from one of the following

or R₁ and R₂, together with the atoms to which they are attached, form afive-membered heterocycle defined by the structure below

In some embodiments, the ¹³C isotopic enrichment factor for ^(d)C is atleast 25 (27.5% ¹³C incorporation at each position), at least 30 (33%¹³C incorporation at each position), at least 35 (38.5% ¹³Cincorporation at each position), at least 40 (44% ¹³C incorporation ateach position), at least 45 (49.5% ¹³C incorporation at each position),at least 50 (55% ¹³C incorporation at each position), at least 55 (60.5%¹³C incorporation at each position), at least 60 (66% ¹³C incorporationat each position), at least 65 (71.5% ¹³C incorporation at eachposition), at least 70 (77% ¹³C incorporation at each position), atleast 75 (82.5% ¹³C incorporation at each position), at least 80 (88%¹³C incorporation at each position), at least 85 (93.5% ¹³Cincorporation at each position), or at least 90 (99% ¹³C incorporationat each position).

In some embodiments, the ¹⁵N isotopic enrichment factor for ^(f)N is atleast 100 (37% ¹⁵N incorporation at each position), at least 110 (40.7%¹⁵N incorporation at each position), at least 120 (44.4% ¹⁵Nincorporation at each position), at least 130 (48.1% ¹⁵N incorporationat each position), at least 140 (51.8% ¹⁵N incorporation at eachposition), at least 150 (55.5% ¹⁵N incorporation at each position), atleast 160 (59.2% ¹⁵N incorporation at each position), at least 170(62.9% ¹⁵N incorporation at each position), at least 180 (66.6% ¹⁵Nincorporation at each position), at least 190 (70.3% ¹⁵N incorporationat each position), at least 200 (74% ¹⁵N incorporation at eachposition), at least 210 (77.7% ¹⁵N incorporation at each position), atleast 220 (81.4% ¹⁵N incorporation at each position), at least 230(85.1% ¹⁵N incorporation at each position), at least 240 (88.8% ¹⁵Nincorporation at each position), at least 250 (92.5% ¹⁵N incorporationat each position), at least 260 (96.2% ¹⁵N incorporation at eachposition), or at least 265 (98.05% ¹⁵N incorporation at each position).

Optionally, ^(a)C, ^(b)C, and/or ^(c)C can also be labeled with a stableisotope. In some cases, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, and ^(c)C can be at least 25 (27.5% ¹³C incorporation at eachposition), at least 30 (33% ¹³C incorporation at each position), atleast 35 (38.5% ¹³C incorporation at each position), at least 40 (44%¹³C incorporation at each position), at least 45 (49.5% ¹³Cincorporation at each position), at least 50 (55% ¹³C incorporation ateach position), at least 55 (60.5% ¹³C incorporation at each position),at least 60 (66% ¹³C incorporation at each position), at least 65 (71.5%¹³C incorporation at each position), at least 70 (77% ¹³C incorporationat each position), at least 75 (82.5% ¹³C incorporation at eachposition), at least 80 (88% ¹³C incorporation at each position), atleast 85 (93.5% ¹³C incorporation at each position), or at least 90 (99%¹³C incorporation at each position).

Optionally, ^(e)N can also be labeled with a stable isotope. In somecases, the ¹⁵N isotopic enrichment factor for ^(e)N is at least 100 (37%¹⁵N incorporation at each position), at least 110 (40.7% ¹⁵Nincorporation at each position), at least 120 (44.4% ¹⁵N incorporationat each position), at least 130 (48.1% ¹⁵N incorporation at eachposition), at least 140 (51.8% ¹⁵N incorporation at each position), atleast 150 (55.5% ¹⁵N incorporation at each position), at least 160(59.2% ¹⁵N incorporation at each position), at least 170 (62.9% ¹⁵Nincorporation at each position), at least 180 (66.6% ¹⁵N incorporationat each position), at least 190 (70.3% ¹⁵N incorporation at eachposition), at least 200 (74% ¹⁵N incorporation at each position), atleast 210 (77.7% ¹⁵N incorporation at each position), at least 220(81.4% ¹⁵N incorporation at each position), at least 230 (85.1% ¹⁵Nincorporation at each position), at least 240 (88.8% ¹⁵N incorporationat each position), at least 250 (92.5% ¹⁵N incorporation at eachposition), at least 260 (96.2% ¹⁵N incorporation at each position), orat least 265 (98.05% ¹⁵N incorporation at each position).

In some embodiments, m can be at least 1 (e.g., at least 5, at least 10,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100). In some embodiments,m can be 108 or less (e.g., 100 or less, 90 or less, 80 or less, 70 orless, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 orless, or 5 or less). m can be an integer ranging from any of the minimumvalues described above to any of the maximum values described above. Forexample, in some embodiments, m can be an integer from 1 to 108 (e.g.,from 1 to 100, from 1 to 50, from 1 to 30, or from 1 to 10).

In some embodiments, n can be at least 1 (e.g., at least 5, at least 10,at least 20, at least 30, at least 40). In some embodiments, n can be 46or less (e.g., 40 or less, 30 or less, 20 or less, 10 or less, or 5 orless). n can be an integer ranging from any of the minimum valuesdescribed above to any of the maximum values described above. Forexample, in some embodiments, n can be an integer from 1 to 46 (e.g.,from 1 to 40, from 1 to 30, from 1 to 20, or from 1 to 10).

In some embodiments, the sum of m and n can be at least 1 (e.g., atleast 5, at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 110, at least 120, at least 130, at least 140, at least 150). Insome embodiments, the sum of m and n can be 154 or less (e.g., 150 orless, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less,90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less,30 or less, 20 or less, 10 or less, or 5 or less). The sum of m and ncan range from any of the minimum values described above to any of themaximum values described above. For example, in some embodiments, thesum of m and n can be from 1 to 154 (e.g., from 1 to 150, from 1 to 100,from 1 to 50, from 1 to 30, from 1 to 10, from 5 to 150, from 5 to 100,from 5 to 50, from 5 to 30, or from 5 to 10).

The composition can be, for example, a solution of the isotopicallylabeled peptide in a solvent. Non-limiting examples of solvents includealcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethylacetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethersand glycol ether esters; tetrahydrofuran; dimethylformamide;acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g.,PBS buffer), and combinations thereof. In certain examples, thecomposition can comprise an aqueous solution of the peptide.

In some embodiments, the isotopically labeled peptide can comprise atleast 0.5% by weight (e.g., at least 1% by weight, at least 1.5% byweight, at least 2% by weight, at least 2.5% by weight, at least 3% byweight, at least 3.5% by weight, at least 4% by weight, at least 4.5% byweight, or at least 1% by weight of the composition.

Also provided are compositions comprising an isotopically labeledpolypeptide that includes one or more β-N-methylamino-L-alanine (BMAA)residues (e.g., one or more BMAA residues that are not isotopicallylabeled) and one or more additional amino acid residues that are labeledwith one or more stable isotopes (¹³C, ¹⁵N, and/or ¹⁸O). In someembodiments, each residue labeled with one or more stable isotopes inthe polypeptide includes at least two (e.g., at least four, at leastfive, at least six, or more) stable isotopes. For example, at least two(e.g., at least four, at least five, at least six, or more) of thecarbon, nitrogen, and/or oxygen atoms in the residue can be isotopicallylabeled with a stable isotope. In some cases, at least two (e.g., atleast four, at least five, at least six, or more) of the carbon and/ornitrogen atoms in the residue can be isotopically labeled with a stableisotope. The isotopically labeled polypeptide can comprise at least 0.5%by weight of the composition.

In some embodiments, the polypeptide can include a single isotopicallylabeled residue. In other embodiments, the polypeptide can include twoor more isotopically labeled residues (e.g., three or more isotopicallylabeled residues, four or more isotopically labeled residues, five ormore isotopically labeled residues, or ten or more isotopically labeledresidues). In some embodiments, the polypeptide can include a singleBMAA residue. In other embodiments, the polypeptide can include two ormore BMAA residues (e.g., three or more BMAA residues, four or more BMAAresidues, five or more BMAA residues, or ten or more BMAA residues).

In certain embodiments, the isotopically labeled polypeptide can be apeptide that includes one or more BMAA residues and a terminal aminoacid residue (e.g., a terminal arginine residue) labeled with one ormore stable isotopes (¹³C, ¹⁵N, and/or ¹⁸O).

The composition can be, for example, a solution of the isotopicallylabeled peptide in a solvent. Non-limiting examples of solvents includealcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethylacetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethersand glycol ether esters; tetrahydrofuran; dimethylformamide;acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g.,PBS buffer), and combinations thereof. In certain examples, thecomposition can comprise an aqueous solution of the peptide.

In some embodiments, the isotopically labeled peptide can comprise atleast 0.5% by weight (e.g., at least 1% by weight, at least 1.5% byweight, at least 2% by weight, at least 2.5% by weight, at least 3% byweight, at least 3.5% by weight, at least 4% by weight, at least 4.5% byweight, or at least 1% by weight of the composition.

The peptides described above can be above can be prepared using avariety of methods known in the art. For example, peptides can beprepared using the isotopically labeled compounds described herein viasolid phase peptide synthesis. The proteins and peptides described abovecan also be prepared by chemical derivatization of one or more residueswithin the protein and peptide. For example, a protein or peptide havinga isotopically labeled β-N-methylamino-L-alanine (BMAA) residue can beprepared from a protein or peptide that includes a phosphoserineresidue. The protein or peptide can be reacted to convert thephosphoserine residue to an α,β-unsaturated amino acid residue. Onceactivated, the α,β-unsaturated amino acid residue can be reacted withmethylamine (e.g., methylamine that is isotopically enriched with one ormore stable isotopes), which undergoes a Michael-type addition to affordan isotopically labeled BMAA residue. This can involve, for example,reaction of the protein or peptide with methylamine-HCl (e.g., 1.0 M¹³C/¹⁵N-labeled methylamine) and Ba(OH)₂ (e.g., 0.1 M Ba(OH)₂) inwater/DMSO/EtOH (2:2:1) at basic pH (pH 12.5) and elevated temperature(e.g., 37° C.). Once complete, the reaction can be quenched with acid(e.g., acetic acid).

Full length natural or stable isotope labeled proteins comprising stableisotope labeled BMAA residues (e.g., incorporated at one or morespecific sites within the protein) can be prepared by first preparing aphosphoserine-containing protein using an amber stop codon and a tRNAsynthetase engineered to incorporate phosphoserine within the desiredprotein. See, for example, Rogerson, D. T. et al. “Efficient geneticencoding of phosphoserine and its nonhydrolyzable analog.” Nat. Chem.Biol., 2015, 7: 496-503. The protein can then be isolated, and thephosphoserine can be chemically converted into BMAA using the strategydescribed above.

Further provided herein are isotopically labeled reagents, includingisotopically labeled small molecules and peptides, that can be used todetect and/or quantify β-N-methylamino-L-alanine (BMAA) in a proteinselected from the validated targets in Table 6. Using these novelreagents, the inventors have identified the incorporation of BMAA intonumerous proteins in samples from patients suffering from amyotrophiclateral sclerosis (ALS) and from control samples. The reagents can beused as stable isotope labeled standards in analytical methods,including in conjunction with mass spectrometry, to detect and/orquantify BMAA in a sample, such as a protein sample from a subject.

In one aspect, provided herein is a polypeptide comprising a sequencethat is at least 85% identical a sequence selected from SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49,wherein at least one amino acid comprises a BMAA residue which isisotopically labeled and is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least 100.

In one aspect, provided herein is a polypeptide comprising a sequenceselected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, or SEQ ID NO:49, wherein at least one amino acid comprisesa BMAA residue which is isotopically labeled and is defined by theformula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least 100.

In one aspect, provided herein is a polypeptide comprising a sequencethat is at least 85% identical a sequence selected from SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75,wherein at least one amino acid comprises a BMAA residue which isisotopically labeled and is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least 100.

In one aspect, provided herein is a polypeptide comprising a sequenceselected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73,SEQ ID NO:74, or SEQ ID NO:75, wherein at least one amino acid comprisesa BMAA residue which is isotopically labeled and is defined by theformula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least 100.

Methods

The compounds, peptides, proteins, and compositions described herein canbe used detect and/or quantify BMAA in a SOD1 protein from a biologicalspecimen (e.g., a sample from a patient suffering from neurodegenerativedisease such as ALS), to accurately monitor BMAA exposure, to directtherapies, and in clinical diagnosis and prognosis.

For example, the isotopically labeled reagents and compositionsdescribed herein can be used in a variety of analytical methods todetect and/or quantify BMAA, such as to detect and/or quantify BMAA in abiological specimen or sample such as a protein sample. The isotopicallylabeled compounds described herein can be used to quantify free BMAA(e.g., quantify free BMAA in an environmental sample or biologicalsample), quantify total levels of BMAA in a protein sample (for example,a SOD1 protein sample), and/or to quantify protein-specific BMAAincorporation (e.g., by upstream purification of the protein of interestprior to analysis). The isotopically labeled compounds described hereincan also be utilized in a stable isotope labeling by amino acids in cellculture (SILAC) alone or in combination with other stable isotopelabeled amino acids. The isotopically labeled compounds described hereinprovide many analytical advantages over potential alternatives, such asdeuterated BMAA, which does not co-elute and can undergohydrogen-deuterium exchange in-solution or in the gas phase,significantly impacting identification and quantitation.

In one aspect, provided herein is a method for detectingβ-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising (a)purifying SOD1 protein from a biological specimen to provide a purifiedSOD1 protein sample, (b) spiking the purified SOD1 protein sample with adefined amount of an isotopically labeled compound defined by theformula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and(d) measuring BMAA levels in the SOD1 protein sample by isotope dilutionanalysis.

In one embodiment, at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.In one embodiment, at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.In one embodiment, at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,and ^(f)N are isotopically labeled with a stable isotope. In oneembodiment, at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and^(f)N are isotopically labeled with a stable isotope. In one embodiment,^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and ^(f)N are isotopically labeledwith a stable isotope.

In one embodiment, the isotopically labeled compound is defined by theformula below

wherein R represents hydrogen or an amine protecting group; wherein the¹³C isotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is atleast 25; and wherein the ¹⁵N isotopic enrichment factor for ^(e)N and^(f)N is at least 100.

In one embodiment, the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C,^(c)C, and ^(d)C is at least 80 and the ¹⁵N isotopic enrichment factorfor ^(e)N and ^(f)N is at least 200. In one embodiment, R is hydrogen.In one embodiment, R represents a 9-fluorenylmethyloxycarbonyl group.

On one embodiment, the purified SOD1 protein sample is hydrolyzed intoamino acids for free BMAA analysis of SOD1.

In one aspect, provided herein is a method for detectingβ-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising: (a)purifying SOD1 protein from a biological specimen to provide a purifiedSOD1 protein sample; (b) spiking the purified SOD1 protein sample with adefined amount of a polypeptide, wherein the polypeptide includes one ormore isotopically labeled β-N-methylamino-L-alanine (BMAA) residues,wherein each of the one or more isotopically labeled BMAA residues isisotopically labeled with one or more stable isotopes; to provide aBMAA-spiked sample; (c) analyzing the BMAA-spiked sample by massspectrometry; and (d) measuring BMAA levels in the SOD1 protein sampleby isotope dilution analysis.

In one embodiment, each of the one or more isotopically labeled BMAAresidues is isotopically labeled with two or more stable isotopes. Inone embodiment, each of the one or more isotopically labeled BMAAresidues is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least 100.

In one embodiment, the ¹³C isotopic enrichment factor for ^(d)C is atleast 80 and the ¹⁵N isotopic enrichment factor for ^(f)N is at least200. In one embodiment, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, and ^(c)C is at least 25. In one embodiment, the ¹³C isotopicenrichment factor for ^(a)C, ^(b)C, and ^(c)C is at least 80. In oneembodiment, the ¹⁵N isotopic enrichment factor for ^(e)N is at least100. In one embodiment, the ¹⁵N isotopic enrichment factor for ^(e)N isat least 200.

In one embodiment, the polypeptide comprises a sequence is at least 85%identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprisesa sequence is at least 90% identical to SEQ ID NO: 1. In one embodiment,the polypeptide comprises a sequence is at least 95% identical to SEQ IDNO: 1. In one embodiment, the polypeptide comprises a sequence is atleast 99% identical to SEQ ID NO: 1. In one embodiment, the polypeptidecomprises a sequence is identical to SEQ ID NO: 1. In one embodiment,the polypeptide comprises a sequence is at least 85% identical to SEQ IDNO: 2. In one embodiment, the polypeptide comprises a sequence is atleast 90% identical to SEQ ID NO: 2. In one embodiment, the polypeptidecomprises a sequence is at least 95% identical to SEQ ID NO: 2. In oneembodiment, the polypeptide comprises a sequence is at least 99%identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprisesa sequence is identical to SEQ ID NO: 2. In one embodiment, the purifiedSOD1 protein sample is digested with trypsin.

In one aspect, provided herein is a method of detecting or predictingamyotrophic lateral sclerosis in a subject, comprising (a) purifyingSOD1 protein from a biological specimen from a subject to provide apurified SOD1 protein sample; (b) spiking the purified SOD1 proteinsample with a defined amount of an isotopically labeled compound definedby the formula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d)measuring BMAA level in the SOD1 protein sample by isotope dilutionanalysis; and (e) identifying the subject for the presence or risk ofamyotrophic lateral sclerosis if the BMAA level in the SOD1 protein isgreater than a normal reference value.

In one embodiment, at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.In one embodiment, at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.In one embodiment, at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,and ^(f)N are isotopically labeled with a stable isotope. In oneembodiment, at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and^(f)N are isotopically labeled with a stable isotope. In one embodiment,^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and ^(f)N are isotopically labeledwith a stable isotope. In one embodiment, the isotopically labeledcompound is defined by the formula below

wherein R represents hydrogen or an amine protecting group; wherein the¹³C isotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is atleast 25; and wherein the ¹⁵N isotopic enrichment factor for ^(e)N and^(f)N is at least 100. In one embodiment, the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is at least 80 and the ¹⁵Nisotopic enrichment factor for ^(e)N and ^(f)N is at least 200. In oneembodiment, R is hydrogen. In one embodiment, R represents a9-fluorenylmethyloxycarbonyl group.

In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, crotonaldehyde, glyoxalderived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehydederived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation andnitrosylation. In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, and crotonaldehyde.

In one embodiment, the subject being identified for the presence or riskof amyotrophic lateral sclerosis comprises a BMAA level in the SOD1protein that is at least 10% greater (e.g. at least 10% greater, atleast 20% greater, at least 50% greater, at least 100% greater, or more)than the normal reference value.

In one aspect, provided herein is a method of detecting or predictingamyotrophic lateral sclerosis in a subject, comprising (a) purifyingSOD1 protein from a biological specimen from a subject to provide apurified SOD1 protein sample; (b) spiking the purified SOD1 proteinsample with a defined amount of a polypeptide, wherein the polypeptideincludes one or more isotopically labeled β-N-methylamino-L-alanine(BMAA) residues, wherein each of the one or more isotopically labeledBMAA residues is isotopically labeled with one or more stable isotopes,to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample bymass spectrometry; (d) measuring BMAA level in the SOD1 protein sampleby isotope dilution analysis; and (e) identifying the subject for thepresence or risk of amyotrophic lateral sclerosis if the BMAA level inthe SOD1 protein is greater than a normal reference value.

In one embodiment, each of the one or more isotopically labeled BMAAresidues is isotopically labeled with two or more stable isotopes. Inone embodiment, each of the one or more isotopically labeled BMAAresidues is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least 100. Inone embodiment, the ¹³C isotopic enrichment factor for ^(d)C is at least80 and the ¹⁵N isotopic enrichment factor for ^(f)N is at least 200. Inone embodiment, the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C, and^(c)C is at least 25. In one embodiment, the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, and ^(c)C is at least 80. In one embodiment,the ¹⁵N isotopic enrichment factor for ^(e)N is at least 100. In oneembodiment, the ¹⁵N isotopic enrichment factor for ^(e)N is at least200.

In one embodiment, the polypeptide comprises a sequence is at least 85%identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprisesa sequence is at least 90% identical to SEQ ID NO: 1. In one embodiment,the polypeptide comprises a sequence is at least 95% identical to SEQ IDNO: 1. In one embodiment, the polypeptide comprises a sequence is atleast 99% identical to SEQ ID NO: 1. In one embodiment, the polypeptidecomprises a sequence is identical to SEQ ID NO: 1. In one embodiment,the polypeptide comprises a sequence is at least 85% identical to SEQ IDNO: 2. In one embodiment, the polypeptide comprises a sequence is atleast 90% identical to SEQ ID NO: 2. In one embodiment, the polypeptidecomprises a sequence is at least 95% identical to SEQ ID NO: 2. In oneembodiment, the polypeptide comprises a sequence is at least 99%identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprisesa sequence is identical to SEQ ID NO: 2.

In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, crotonaldehyde, glyoxalderived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehydederived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation andnitrosylation. In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, and crotonaldehyde.

In one aspect, provided herein is a method of preventing or treatingamyotrophic lateral sclerosis in a subject, comprising (a) purifyingSOD1 protein from a biological specimen from a subject to provide apurified SOD1 protein sample; (b) spiking the purified SOD1 proteinsample with a defined amount of an isotopically labeled compound definedby the formula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d)measuring BMAA levels in the SOD1 protein sample by isotope dilutionanalysis; (e) identifying the subject for the presence or risk ofamyotrophic lateral sclerosis if the BMAA levels in the SOD1 protein isgreater than a normal reference value; and (f) administering to thesubject L-serine in an amount sufficient to prevent or treat theamyotrophic lateral sclerosis.

In one embodiment, at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.In one embodiment, at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.In one embodiment, at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,and ^(f)N are isotopically labeled with a stable isotope. In oneembodiment, at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and^(f)N are isotopically labeled with a stable isotope. In one embodiment,^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and ^(f)N are isotopically labeledwith a stable isotope. In one embodiment, the isotopically labeledcompound is defined by the formula below

wherein R represents hydrogen or an amine protecting group; wherein the¹³C isotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is atleast 25; and wherein the ¹⁵N isotopic enrichment factor for ^(e)N and^(f)N is at least 100. In one embodiment, the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is at least 80 and the ¹⁵Nisotopic enrichment factor for ^(e)N and ^(f)N is at least 200. In oneembodiment, R is hydrogen. In one embodiment, R represents a9-fluorenylmethyloxycarbonyl group.

In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, crotonaldehyde, glyoxalderived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehydederived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation andnitrosylation. In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, and crotonaldehyde.

In one aspect, provided herein is a method of preventing or treatingamyotrophic lateral sclerosis in a subject, comprising (a) purifyingSOD1 protein from a biological specimen from a subject to provide apurified SOD1 protein sample; (b) spiking the purified SOD1 proteinsample with a defined amount of a polypeptide, wherein the polypeptideincludes one or more isotopically labeled β-N-methylamino-L-alanine(BMAA) residues, wherein each of the one or more isotopically labeledBMAA residues is isotopically labeled with one or more stable isotopes,to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample bymass spectrometry; (d) measuring BMAA levels in the SOD1 protein sampleby isotope dilution analysis; (e) identifying the subject for thepresence or risk of amyotrophic lateral sclerosis if the BMAA levels inthe SOD1 protein is greater than a normal reference value; and (f)administering to the subject L-serine in an amount sufficient to preventor treat the amyotrophic lateral sclerosis.

In one embodiment, each of the one or more isotopically labeled BMAAresidues is isotopically labeled with two or more stable isotopes. Inone embodiment, each of the one or more isotopically labeled BMAAresidues is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least 100. Inone embodiment, at least 80 and the ¹⁵N isotopic enrichment factor for^(f)N is at least 200. In one embodiment, the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, and ^(c)C is at least 25. In one embodiment,the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C, and ^(c)C is atleast 80. In one embodiment, the ¹⁵N isotopic enrichment factor for^(e)N is at least 100. In one embodiment, the ¹⁵N isotopic enrichmentfactor for ^(e)N is at least 200. In one embodiment, the polypeptidecomprises a sequence is at least 85% identical to SEQ ID NO: 1. In oneembodiment, the polypeptide comprises a sequence is at least 90%identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprisesa sequence is at least 95% identical to SEQ ID NO: 1. In one embodiment,the polypeptide comprises a sequence is at least 99% identical to SEQ IDNO: 1. In one embodiment, the polypeptide comprises a sequence isidentical to SEQ ID NO: 1. In one embodiment, the polypeptide comprisesa sequence is at least 85% identical to SEQ ID NO: 2. In one embodiment,the polypeptide comprises a sequence is at least 90% identical to SEQ IDNO: 2. In one embodiment, the polypeptide comprises a sequence is atleast 95% identical to SEQ ID NO: 2. In one embodiment, the polypeptidecomprises a sequence is at least 99% identical to SEQ ID NO: 2. In oneembodiment, the polypeptide comprises a sequence is identical to SEQ IDNO: 2.

In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, crotonaldehyde, glyoxalderived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehydederived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation andnitrosylation. In one embodiment, the SOD1 protein further comprises apost-translational modification selected from the group consisting of aphospho group, acetyl group, glutathione, and crotonaldehyde.

In some embodiments, after the detection of BMAA in SOD1, the patientmay be treated with L-serine. In some embodiments, after the detectionof BMAA in SOD1, the patient may be treated with L-serine, or aprecursor, derivative or conjugate of L-serine.

While there is no known cure for ALS, the FDA approved the use ofriluzole, an anti-glutamate agent, to help slow down the progression ofthe disease (See U.S. Pat. No. 4,826,860). Riluzole is believed toreduce damage to motor neurons by decreasing the release of glutamate.Clinical trials with ALS patients showed that riluzole prolongs survivalby several months, mainly in those with difficulty swallowing. The drugcan also extend the time before an individual needs ventilation support.Other anti-glutamate agents, such as Talampanel and Memantine, have alsobeen proposed as potential treatments for ALS. Currently, physicians canalso prescribe medications to help reduce symptoms of ALS, such asfatigue, ease muscle cramps, control spasticity, and reduce excesssaliva and phlegm. In some embodiments, after the detection of BMAA inSOD1, the patient may be treated with riluzole. In some embodiments,after the detection of BMAA in SOD1, the patient may be treated withtalampanel or memantine.

Recently, it has been proposed to treat ALS with stem cells (See U.S.Pat. Nos. 5,968,829 and 8,765,119). It is thought that stem cellsinjected into the spinal cord of patients with ALS can not only matureinto new nerve and spinal cells, but also release chemicals to protectexisting nerve cells and their connections. Other proposed treatmentsinclude anti-apoptosis agents, such as Minocyclin, and anti-oxidativeagents, such as Tamoxifen. Minocycline (see U.S. Patent App. Pub. No.20150190415) is currently in a large Phase III clinical trial. In someembodiments, after the detection of BMAA in SOD1, the patient may betreated with stem cell therapy. In some embodiments, after the detectionof BMAA in SOD1, the patient may be treated with tamoxifen orminocycline.

In one embodiment, the ALS is sporadic ALS. In one embodiment, the ALSis familial ALS.

The SOD1 protein, the BMAA-spiked sample, or a combination thereof canbe prepared for analysis by mass spectrometry by a method comprisingchemical reactions with flight enhancers, chemical fragmentation,enzymatic digestion, purification, or a combination thereof. In someembodiments, the isotopically labeled compounds described herein can beused to detect and quantify BMAA obtained from the hydrolytic cleavageof amino acids from a target protein as well as for identification andquantification of BMAA incorporated into proteins at the peptide andprotein level utilizing isotope dilution mass spectrometry.

The proteins and peptides described herein can be used as diagnosticmarkers, to monitor exposure to BMAA, and/or to identify diseaserelevant and functionally important proteins in which BMAA has beenincorporated in specific sequence locations. The proteins and peptidesdescribed herein can be utilized in a protein-cleavage-isotope dilutionworkflow to confirm the primary structure, and to accurately andprecisely quantify BMAA incorporated into peptides produced via chemicalor enzymatic digestion of specific proteins.

These proteins and peptides can also be employed in separation schemes,followed by intact mass spectrometry or peptide level detection andquantification through chemical or proteolytic digestion. These proteinsand peptides can also be used to produce antibodies, aptamers and/orother affinity reagents which can be utilized for other diagnostic toolsand applications. These proteins and peptides can also be used inbiophysical studies, providing a method for studying the effect of thisnon-protein amino acid incorporation in proteins. In some embodiments,antibodies can be produced that have affinity for the SOD1 protein, orfragment of the SOD1 protein, containing a BMAA residue. In oneembodiment, the BMAA residue is substituted at the Ser107 position inSOD1. In one embodiment, antibodies can be produced that have affinityfor the SOD1 protein, or fragment of the SOD1 protein, containing a BMAAresidue, wherein the antibodies do not have significant affinity for thecorresponding SOD1 protein lacking the BMAA residue. In someembodiments, the antibody recognizes an epitope of SOD1 comprising BMAAat the Ser107 position. In some embodiments, the antibody recognizes anepitope of SOD1 that does not comprise BMAA at the Ser107 position.

The methods herein can also be used to detect β-N-methylamino-L-alanine(BMAA) in additional proteins in a patient sample. In one aspect,provided herein is a method for detecting β-N-methylamino-L-alanine(BMAA) in a protein, comprising (a) purifying the protein from abiological specimen to provide a purified protein sample, (b) spikingthe purified protein sample with a defined amount of an isotopicallylabeled compound defined by the formula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and(d) measuring BMAA levels in the protein sample by isotope dilutionanalysis.

In one aspect, provided herein is a method for detectingβ-N-methylamino-L-alanine (BMAA) in a protein, comprising: (a) purifyingthe protein from a biological specimen to provide a purified proteinsample; (b) spiking the purified protein sample with a defined amount ofa polypeptide, wherein the polypeptide includes one or more isotopicallylabeled β-N-methylamino-L-alanine (BMAA) residues, wherein each of theone or more isotopically labeled BMAA residues is isotopically labeledwith one or more stable isotopes; to provide a BMAA-spiked sample; (c)analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuringBMAA levels in the protein sample by isotope dilution analysis.

In another aspect, provided herein is a method of detecting orpredicting amyotrophic lateral sclerosis in a subject, comprising (a)purifying a protein from a biological specimen from a subject to providea purified protein sample; (b) spiking the purified protein sample witha defined amount of an isotopically labeled compound defined by theformula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d)measuring BMAA level in the protein sample by isotope dilution analysis;and (e) identifying the subject for the presence or risk of amyotrophiclateral sclerosis if the BMAA level in the protein is greater than anormal reference value.

In one aspect, provided herein is a method of preventing or treatingamyotrophic lateral sclerosis in a subject, comprising (a) purifying aprotein from a biological specimen from a subject to provide a purifiedprotein sample; (b) spiking the purified protein sample with a definedamount of an isotopically labeled compound defined by the formula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope, to provide a BMAA-spikedsample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d)measuring BMAA levels in the protein sample by isotope dilutionanalysis; (e) identifying the subject for the presence or risk ofamyotrophic lateral sclerosis if the BMAA levels in the protein isgreater than a normal reference value; and (f) administering to thesubject L-serine in an amount sufficient to prevent or treat theamyotrophic lateral sclerosis.

In still a further aspect, provided herein is a method of preventing ortreating amyotrophic lateral sclerosis in a subject, comprising (a)purifying a protein from a biological specimen from a subject to providea purified protein sample; (b) spiking the purified protein sample witha defined amount of a polypeptide, wherein the polypeptide includes oneor more isotopically labeled β-N-methylamino-L-alanine (BMAA) residues,wherein each of the one or more isotopically labeled BMAA residues isisotopically labeled with one or more stable isotopes, to provide aBMAA-spiked sample; (c) analyzing the BMAA-spiked sample by massspectrometry; (d) measuring BMAA levels in the protein sample by isotopedilution analysis; (e) identifying the subject for the presence or riskof amyotrophic lateral sclerosis if the BMAA levels in the protein isgreater than a normal reference value; and (f) administering to thesubject L-serine in an amount sufficient to prevent or treat theamyotrophic lateral sclerosis.

The proteins or polypeptides used in the methods described herein cancomprise any of the proteins or protein fragments described in Table 6or Table 7 below.

By way of non-limiting illustration, examples of certain embodiments ofthe present disclosure are given below.

EXAMPLES Example 1 Preparation of ¹³C₄ ¹⁵N₂ β-N-Methylamino-L-Alanine

Preparation of Compound 1. To a solution of ¹³C-¹⁵N labeled L-asparaginemonohydrate (95.8 mg, 0.614 mmol) in 10% aqueous Na₂CO₃ (1.6 mL) wasadded 1,4-dioxane (0.9 mL) and the mixture was cooled to 0° C. Benzylchloroformate (130 mg, 0.737 mmol) was then added and the mixture wasallowed to warm to rt overnight. The reaction mixture was poured intowater (4.0 mL), and the mixture was extracted with diethyl ether (×3).The aqueous layer was then acidified with an aqueous solution of 2N HCl(pH=2), and the white solid was filtered to afford 98.5 mg (59%) of theproduct: ¹H NMR (400 MHz, CDCl₃) δ 7.37 (m, 5H), 5.10 (m, 2H), 4.25 (bd,1H, J=132.1 Hz), 2.81 (bd, 1H, J=44.1 Hz), 2.46 (bd, 1H, J=64.6 Hz);ESIMS m/z 273 [M+H]⁺; HRMS m/z calculated for ¹³C₄C₈H₁₄ ¹⁵N₂O₅ [M+Na]⁺295.0870, found 295. 0867.

Preparation of Compound 2. To a slurry of N²-benzyloxycarbonylasparagine(98.5 mg, 0.362 mmol) in ethyl acetate (0.89 mL), acetonitrile (0.96mL), and water (0.46 mL) was added iodosobenzene diacetate (0.166 g,0.507 mmol) at 15° C. and the mixture was stirred for 30 min at 15° C.The reaction mixture was then allowed to warm to rt and stirred untilcompletion (4 h). The mixture was cooled to 5° C., and the product wascollected, washed with ethyl acetate, and dried in vacuo to afford 33.9mg (39%) of the product as a white solid: ESIMS m/z 244 [M+H]⁺. ¹H NMR(400 MHz, CDCl₃) δ 8.03 (brs, 1H), 7.85 (brs, 2H), 7.40 (s, 5H), 5.07(s, 2H), 4.30 (bd, 1H, J=139.2 Hz), 3.31 (bd, 1H, J=83.9 Hz), 3.31 (bd,1H, J=83.9 Hz), 2.95 (bd, 1H, J=87.9 Hz); ESIMS m/z 244 [M+H]⁺; HRMS m/zcalculated for ¹³C₃C₈H₁₄ ¹⁵N₂O₄ [M+H]⁺ 244.1068, found 244.1066.

Preparation of Compound 3. To a suspension of compound 3 (33.9 mg, 0.139mmol) in methanol (0.60 mL) was added Et₃N (42.3 mg, 0.416 mmol) andbenzaldehyde (29.6 mg, 0.278 mmol) at rt, and the mixture was stirredfor 30 min. The reaction mixture was cooled to 0° C., followed by theaddition of NaBH₄ (16.0 mg, 0.416 mmol). The mixture was then stirredfor an additional 15 min at 0° C., and concentrated under reducedpressure. The residue was then dissolved in 0.1 M aqueous solution ofNaOH, and extracted with diethyl ether (×3). The aqueous layer was thenacidified with an aqueous solution of 10% hydrochloric acid, and theresultant white precipitate was filtered to afford 27.2 mg of theproduct. The white solid was dissolved in methanol (0.27 mL), and asolution of 35% aqueous solution of formaldehyde (18.2 μL, 0.244 mmol)was added. The reaction was stirred for an additional 15 min, and cooledto 0° C. NaBH₄ (9.32 mg, 0.244 mmol) was then added, and the mixture wasstirred for 15 min. Upon completion, the mixture was concentrated underreduced pressure, and the crude residue was dissolved in water,acidified (pH=6) with a 1 M aqueous solution of HCl, extracted withCHCl₃, dried (MgSO₄), and concentrated under reduced pressure to affordthe crude product. The crude product was triturated with diethyl etherto afford 28.3 mg (100%) of the product as a white solid: ¹H NMR (400MHz, CDCl₃) δ 7.18 (s, 10H), 6.38 (d, 1H, J=92.5 Hz), 5.09 (d, 1H,J=16.5 Hz), 4.88 (d, 1H, J=15.4 Hz), 4.19 (d, 1H, J=83.9 Hz), 3.58 (d,1H, J=13.2 Hz), 3.48 (d, 1H, J=16.1 Hz), 3.01 (s, 1H), 2.67 (s, 1H),2.12 (s, 3H); ESIMS m/z 348 [M+H]⁺; HRMS m/z calculated for ¹³C₃C₁₆H₂₂¹⁵N₂O₄ [M+H]⁺ 348.1694, found 348.1690.

Preparation of Compound ¹³C₄ ¹⁵N₂ β-N-Methylamino-L-Alanine. To adegassed solution of compound 3 (28.3 mg, 0.0814 mmol) in methanol (0.8mL) was added Pd/C (8.66 mg, 0.00813 mmol), and the mixture was furtherdegassed for an additional 5 min. The mixture was then saturated with H₂gas and stirred under a H₂ atmosphere overnight. The Pd/C was filteredthrough Celite®, and washed with methanol. The filtrated wasconcentrated under reduced pressure and the crude product was trituratedwith diethyl ether to afford 7.7 mg (77%) of the product as a whitesolid: ESIMS m/z 146 [M+Na]⁺; HRMS m/z calculated for ¹³C₃CH₁₀ ¹⁵N₂O₂[M+H]⁺ 124.0856, found 124.0856. This final product was furthercharacterized by MS/MS to confirm the exact structure (FIG. 14).

¹³C₄ ¹⁵N₂ β-N-methylamino-L-alanine

A well-characterized, highly phosphorylated protein, Beta-Casein, wasutilized as a positive control. The intact protein was reacted withmethylamine under the same conditions for peptide synthesis, convertingphosphoserines to BMAA. Purified SOD1 from 3 patients with sporadic ALSand 3 healthy controls were washed on a 10 kDa FASP filter (Millipore)and concentrated to 50 μL. 20 μg of protein was hydrolyzed using 50 of6N HCl and incubating at 110° C. for 18 hours. 5 μL of 60.9 mM SIL BMAAwas spiked into the samples post-hydrolysis. Samples were then dried andresuspended in 100 μL of 0.001% Zwittergent 3-16. Direct infusion ESIMS/MS of SIL BMAA confirmed the location of isotope incorporation. AZipChip (908 Devices) capillary electrophoresis was utilized to separateBMAA prior to electrospray ionization mass spectrometry. Accurate intactmass and migration time of the SIL reagent was used to identify BMAA.

The peptide sequence DGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO:17) withhighlighted (bold and underlined) Serine phosphorylated,carbamidomethylated Cysteine and ¹³C₆ ¹⁵N₄ isotopically labeled Argininewas obtained from New England Peptide. This sequence was validated byaccurate intact mass (FIG. 2) and MS/MS (FIG. 3) which allowed for sitespecific confirmation of phosphorylation. A solution of water, DMSO,EtOH solution in 2:2:1 mixture containing 0.1M Barium Hydroxide and 1MMethylamine pH 12.5 was used for derivatization of the SIL peptide. 5 (5μg) of peptide was added to 20 μL of derivatization solution andincubated at 37° C. for two hours. The reaction was quenched with 2 μLof Acetic Acid. See FIG. 1 for synthesis reference. The formation ofBMAA at the phosphorylated Serine residue was confirmed by accurateintact mass (FIG. 2) and MS/MS (FIG. 4). Further, the R.T of the BMAApeptide was distinctly different from that of the phosphorylated peptide(FIG. 5), along with co-elution of fragment ions to confirm the identityof this species. The conserved relative abundance of the fragmentsbelonging to the SIL BMAA peptide act as a standard for furtherconfirmation.

Each sample was diluted 2-fold in a denaturing solution of 100 mM DTT(15.43 mg/mL) and 8 M urea. Then the samples were incubated at 56° C.for 30 minutes. After incubation enough alkylation solution, made from 1M iodoacetamide (184.96 mg/mL) and 8 M urea, was added to give eachsample a final iodoacetamide concentration of 200 mM. Then the sampleswere incubated at 37° C. for an hour. The appropriate amount for eachsample was pipetted into an Amicon Ultra-0.1 MWCO-filter unit (10 kDa,Millipore). These were centrifuged for 15 minutes at 14,000×g and 20°C.; when finished the eluent was discarded. The remaining volume wasdiluted with 400 uL of a digestion buffer made of 2 M urea (102.12mg/mL) and 10 mM CaCl_2 (1.11 mg/mL). The samples were centrifuged againfor 15 minutes at 14,000×g and 20° C. This was repeated two more times,making sure to discard the eluent after each run. After the finalcentrifugation the collection tubes were changed and 45 uL of modifiedporcine trypsin reconstituted in 2 M urea and 10 mM CaCl_2 was added andthe samples were incubated at 37° C. overnight. Then the samples werequenched with 50 uL of 1% formic acid (v/v) and 0.001% Zwittergent 3-16and centrifuged for 15 minutes at 14,000×g and 20° C. 400 uL of quenchbuffer was added to the retained volume and centrifuged again for 15minutes at 14,000×g and 20° C. Samples were frozen at −80° C. andlyosphilized in a speedvac. Immediately prior to analysis samples werereconstituted in 90 uL of Zwittergent 3-16. A BMAA peptide dry aliquotof 5 ug was resuspended in 500 uL of Zwittergent. A 45 uL aliquot of theprotein digest was then spiked with 5 uL of BMAA peptide standard forLC-MS analysis.

LC-MS/MS by parallel reaction monitoring was used to isolate, fragmentand perform accurate mass measurements of our target endogenous BMAApeptide and SIL BMAA peptides. Direct inject column configuration on aThermo Easy nano-LC 1000 system coupled to a QExactive High Field massspectrometer was used. Analytical columns were made using 75 um×15 cmPicoFrit columns (New Objective, Woburn, Mass.) which were self-packedwith Kinetex C18 2.6-um particles (phenomenex, Torrance, Calif., USA).The samples were loaded with a 10 uL injection volume of mobile phase A(98% water, 2% acetonitrile, and 0.2% formic acid) with a max pressureof 500 Bar. A 45 minute run from 2% to 30% mobile phase B (98%acetonitrile, 2% water, and 0.2% formic acid) was performed at a flowrate of 300 nL/min. The analysis had the following parameters: a sprayvoltage of +1750.00, capillary temperature of 325° C., a S-lens RF levelof 65.00, a MS/MS resolving power of 15,000, a 1e6 AGC target, a 1,000ms fill time, a 2.5 m/z isolation window with an isolation offset of 1.0m/z, a fixed first mass of 125.0 m/z, and a 20, 30 stepped normalizedcollision energy. There was an inclusion list containing 846.4228 m/zand 843.0867 m/z.

Without the SIL BMAA peptide, correct identification of this peakbecomes very difficult as other mass conflicts are present within 5 ppmof the endogenous BMAA peptide as shown in FIG. 7 where the mostabundant peak is the same sequence does not co-elute with the SILpeptide. When examining the correct retention time, the intact massappears to be present within 1.2 ppm mass accuracy and the correctcharge state (+3) is also identified (FIG. 8). Moreover, this peakco-elutes with our SIL peptide as shown in FIG. 9. Fragment ionscorresponding to the endogenous peptide with BMAA at the expectedlocation could be identified at the same retention time (FIG. 13).

Example 2 NanoLC MS and MS/MS Characterization of SOD1 in Human ClinicalALS

Samples for analysis were obtained from control patients (n=10) and ALSpatients (n=7). The SOD1 protein was purified from plasma in each ofthese patient samples. The SOD1 protein can be purified by one of skillin the art (for example, purification using an antibody directed to SOD1or using a protein that has binding affinity for SOD1). In someexperiments, the intact SOD1 protein was analyzed, while in otherexperiments, the SOD1 protein was digested with trypsin before furtheranalysis (See FIG. 10).

For the liquid chromatography experiments, the Thermo Easy nanoLC setupincluded: 75 μm inner diameter 15 cm column utilizing C18 stationaryphase for reversed-phase separation of peptides base on hydrophobicity.The flow rate was 300 nL/min. 2 μL of digested (trypsin) SOD1 containing˜200 ng was injected directly onto the column using a Mobile phase A(98% water, 2% Acetonitrile (ACN), 0.2% formic acid) and a Mobile phaseB (98% ACN, 2% water, 0.2% formic acid). A gradient elution wasperformed from 5%-40%B over 30 min.

Data was collected by Full-MS data dependent MS/MS using a top 20experiment on Q Exactive HF.

For intact protein analysis, 200 ng of undigested protein was injecteddirectly on a 10 cm column using an isocratic (50% A/50% B) elution.Accurate Mass can be obtained by deconvolution with Xtract algorithm.

For the initial SOD1 proteomics search parameters, 69 protein sequencesincluding SOD1 wild-type sequence as well as commonly found samplecontaminants such as keratin and trypsin were identified in proteindatabases. Protein DB is digested in silico using rules for trypticdigestion (Cleaves C-terminal to R/K except when preceded by P) andallowing for potential missed cleavages (up to 3). The search toleranceallowed for 5 ppm MMA (mass measurement accuracy) for peptide, 0.02 Dafor fragment ions. There were two bioinformatics workflows allowing for:

-   1) Dynamic Modifications: Ser→BMAA, Glutathionylation (Cys),    Deamidation (Q,N), Phospho (S,T,Y), Carbamidomethylation (Cys).    Carboxyethyl (MDA), Crotonaldehyde (C,H), 4-HNE (K),    Acrolein/proprionyl (K, T, S), Acetylation (K), Nitrotryptophan (W),    Sulfonylation (C, T, S), Glyoxal derived carboxymethyllysine CML    (K), Crotonaldehyde derived dimethyl-FDP-lysine (K)-   2) SNP Dynamic Modifications: Val→Met, Ala→Val, His→Arg, Thr→Arg,    Gly→Arg, Ser→BMAA (Also searched all other amino acids to BMAA)

Fixed: Carbamidomethylation

Peptides were filtered at 1% False Discovery Rate (FDR).

The total glutathionylation and phosphorylation from 17 patient samples(10 healthy controls and 7 ALS patient samples) were analyzed andresults are shown in Table 1.

TABLE 1 Glutathionylation + Sample Glutathionylation PhosphorylationPhosphorylation HC01 36.31% 14.53% 6.19% HC02 39.17% 21.69% 10.98% HC0330.22% 19.16% 5.39% HC04 33.16% 20.67% 6.10% HC05 28.06% 20.38% 5.19%HC06 34.77% 20.82% 6.54% HC07 41.66% 11.35% 5.12% HC08 35.73% 12.66%3.85% HC09 19.35% 12.97% 1.96% HC10 34.94% 14.09% 4.94% ALS01 28.26%33.91% 9.01% ALS02 0.00% 79.15% 0.00% ALS03 2.34% 72.06% 0.00% ALS0427.75% 24.76% 4.58% ALS06 38.99% 22.18% 8.09% ALS08 38.84% 19.88% 7.32%ALS09 43.98% 20.00% 11.27% p value 0.23 0.02 0.94 *HC = Healthy Controlsample *ALS = Amyotrophic Lateral Sclerosis sample

The post-translational modifications identified from the 17 patientsamples were analyzed and results are shown in Table 2.

TABLE 2 HC MOD ALS MOD Shared Occupancy C111(Glutathione) T2(Phospho)K128(Acetyl) K23(Acetyl) K136(Acetyl) S68(Phospho) K3(Acetyl)K75(Acetyl) K9(Acetyl) T78(Phospho) T39(Phospho) 0.85% HC, 0.89% UNC, p= 0.86 S107(Phospho) K70(Acetyl) C146(Glutathione) T88(Phospho) 0.44%HC, 0.42% UNC, p = 0.94 K91(Acetyl) K122(Acetyl) T54(Phospho) 51% HC,45% UNC, p = 0.84 T58(Phospho) K30(Acetyl) *HC = Healthy Control sample*ALS = Amyotrophic Lateral Sclerosis sample *The amino acid numberingshown in the table does not include the starting methionine.

Known modifications in Table 2 include: C111(Glutathione),S107(Phospho), T2(Phospho), K3(Acetyl), and K122(Acetyl). The othermodifications identified in Table 2 are novel modifications that havenot previously been identified. Modifications identified with highconfidence are shown in FIG. 11, and summarized in Table 3.

TABLE 3 Amino Acid Modification Peptide T2 phosphorylationATKAVCVLKGDGPVQGIINFEQK (SEQ ID NO: 7) K3 acetylationATKAVCVLKGDGPVQGIINFEQK (SEQ ID NO: 7) C6 sulfoAVCVLKGDGPVQGIINFEQK (SEQ ID NO: 8) K9 CMLAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIK (SEQ ID NO: 9) K9 acetylationADDLGKGGNEESTK (SEQ ID NO: 10) K23 CMLAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIK (SEQ ID NO: 9) K30 propionylESNGPVKVWGSIK (SEQ ID NO: 11) W32 nitro ESNGPVKVWGSIK (SEQ ID NO: 11)T39 phosphorylation GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO:12) T54sulfo GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T54 propionylGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) C57 crotonaldehydeGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T58 sulfoGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T88 phosphorylationHVGDLGNVTADK (SEQ ID NO: 13) K91 crotonaldehydeHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ derived dimethyl- ID NO: 14)FDP-lysine K91 CML DEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR(SEQ ID NO: 15) S102/ phosphorylationDFVADVSIEDSVISLSGDHCIIGRTLVVHEK (SEQ ID 105 NO: 16) S107 phosphorylationHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) S107 Ser→BMAADGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 17) H110 crotonaldehydeDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 17) C111 glutathioneHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) C111 crotonaldehydeDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 17) T116 propionylTLVVHEKADDLGK (SEQ ID NO: 18) K122 CMLTLVVHEKADDLGKGGNEESTKTGNAGSR (SEQ ID NO: 19) K122 propionylTLVVHEKADDLGK (SEQ ID NO: 18) K122 acetylationTLVVHEKADDLGK (SEQ ID NO: 18) K128 carboxyethylADDLGKGGNEESTK (SEQ ID NO: 10) K128 propionylADDLGKGGNEESTK (SEQ ID NO: 10) G130 Gly→ArgADDLGKGGNEESTKTGNAGSR (SEQ ID NO: 19) K136 propionylADDLGKGGNEESTKTGNAGSR (SEQ ID NO: 19) K136 acetylationGGNEESTKTGNASGSR (SEQ ID NO: 20) C146 glutathioneTGNAGSRLACGVIGIAQ (SEQ ID NO: 21) *The amino acid numbering shown in thetable does not include the starting methionine.

Modifications identified with medium confidence are shown in FIG. 12 andsummarized in Table 4.

TABLE 4 Amino Acid Modification Peptide S25 propionylESNGPVKVWGSIK (SEQ ID NO: 11) K36 CMLVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 22) T39 propionylGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) H46 carboxyethylGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) H48 carboxyethylGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) A55 HNEVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 22) C57 sulfoGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) C57 HNEGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T58 propionylGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) S59 sulfoGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) S59 propionylGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) H80 carboxyethylHVGDLGNVTADK (SEQ ID NO: 13) K91 HNEHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) K91 acetylationHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) A95 HNEHVGKLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 23) S105 propionylHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) S107 propionylHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) *The amino acidnumbering shown in the table does not include the starting methionine.

The single nucleotide polymorphisms (SNPs) identified from the 17patient samples were analyzed and results are shown in Table 5.

TABLE 5 HC Sequence Variant ALS Sequence Variant Shared Sequence VariantV5(Val−>Met) G10(Gly−>Arg) G130(Gly−>Arg) V103(Val−>Met) G12(Gly−>Arg)V31(Val−>Met) G44(Gly−>Arg) G27(Gly−>Arg) V47(Val−>Met) G51(Gly−>Arg)G82(Gly−>Arg) G72(Gly−>Arg) G127(Gly−>Arg) G16(Gly−>Arg) * The aminoacid numbering shown in the table does not include the startingmethionine.Known single nucleotide polymorphisms (SNPs) in Table 5 include:G12(Gly→Arg). The additional SNPs identified in Table 5 are novel.

Using the novel reagents and methods disclosed herein, the inventorshave identified the incorporation of endogenous BMAA into the SOD1protein in human ALS samples for the first time. Incorporation of BMAAat Serine 107 may act alone, or in combination with other BMAAsubstitutions, SNP(s) and/or post translational modifications (PTMs) onSOD1, other SOD proteins, or in combination with SOD1 binding partners.Additionally, other proteins within the oxidative stress pathway couldincorporate this non-natural amino acid. In the case of SOD1,incorporation of this non-natural amino acid could also reflect tRNAsynthetase mutations or promiscuity.

In some embodiments, the consequences of BMAA incorporation couldinclude, for example, a decrease in the stability of the homodimer, anincrease in the likelihood of SOD1 to aggregate (e.g., trimers which arevery toxic), a decrease in the ability of SOD1 to bind metals, SOD1misfolding, a decrease or alteration of enzymatic activity, and/or tRNAsynthetases have sequence variation altering their specificity.

Example 3 NanoLC MS and MS/MS Characterization of BMAA IncorporationSites in Human Clinical ALS Brain Tissue

Brain tissue samples of 12 matched ALS/healthy control samples wereobtained from Emory University. 100× Halt protease/phosphatase inhibitorwas diluted to 1× in a lysis buffer which comprised 8 M urea and 100 mMammonium bicarbonate at a pH of 7.0. Samples were blended in a BulletBlender with 500 μL of lysis buffer and 750 μg of stainless steel beadsfor 15 minutes at a setting of 3 while refrigerated. A DismembranatorSonicator was used three times for 5 seconds with 15 second breaks. Eachsample was diluted 4-fold with water such that the concentration of Ureawas 2M. The protein concentration of each sample was then determinedusing a Bradford Assay. An appropriate volume of the sample containing250 μg was transferred to a 10 kDa MWCO-filter unit and diluted 2-foldin a denaturing solution made from 100 mM DTT and 8 M urea and heated at56° C. for 30 minutes. The samples were alkylated using 1 Miodoacetamide and 8 M urea to give a final iodoacetamide concentrationof 200 mM and heated at 37° C. for 60 minutes. To concentrate thesamples, they were spun for 15 minutes at 14,000×g and 20° C. Theretained volume was diluted three times with 400 μL of a digestingbuffer of 100 mM ammonium bicarbonate at pH 7.0 and spun for 15 minutesat 14,000×g and 20° C. between each buffer addition. Filters were thentransferred to a clean tube. 2 μg SIL SOD1 surrogate peptide containingBMAA was reconstituted in 500 μL of 0.001% Zwittergent 3-16, then 5 μLwas spiked into each sample. The samples were digested using 45 μL of anenzyme solution containing 100 μg/ml modified trypsin and 100 mMammonium bicarbonate to give a 1:50 enzyme:protein ratio and incubatedovernight at 37° C. 50 μL of a quench buffer made from 1% formic acidand 0.001% Zwittergent 3-16 was added to the filter units which werethen spun for 15 minutes at 14,000×g to elute the peptides. 400 μL ofthe quench buffer was added to the retained volume and spun for 15minutes at 14,000×g using the same collection tube. The samples werelyophilized and kept at −80° C. until analysis. Samples werereconstituted in 100 μL of 0.001% Zwittergent 3-16. 4 μL (approximately0.5 μg) was injected via direct inject column configuration onto aThermo Easy nano-LC 1200 system coupled to a QExactive High Field massspectrometer. Analytical columns were made using 75 um×15 cm PicoFritcolumns (New Objective, Woburn, Mass.) which were self-packed withKinetex C18 2.6-um particles (phenomenex, Torrance, Calif., USA). Thesamples were loaded with a 8 uL injection volume of mobile phase A (98%water, 2% acetonitrile, and 0.2% formic acid) with a max pressure of 600Bar. A 4 hour gradient was performed at 300 nL/min, going from 2% mobilephase B (80% Acetonitrile, 20% water, 0.2% formic acid), to 40%. A top20 data dependent acquisition was performed with the following MSparameters: a spray voltage of +2 kV, capillary temperature of 325° C.,a S-lens RF level of 65.00, an MS resolving power of 120 k and an MS/MSresolving power of 15,000, a 3e6 MS AGC target with a max fill time of50 ms and 1E5 MS/MS AGC with a max fill time of 30 ms and an intensitythreshold of 3.3E4. The data was processed through proteome discoverer2.0 and searched using the Uniprot human proteome FASTA database.Protein DB was digested in silico using rules for tryptic digestion(Cleaves C-terminal to R/K except when preceded by P) and allowing forpotential missed cleavages (up to 3). The search tolerance allowed for 5ppm MMA (mass measurement accuracy) for peptide, 0.02 Da for fragmentions. The following variable modifications were searched:

Serine→BMAA, Carbamidomethylation (Cys), Crotonaldehyde (C,H), andAspartic acid→Glutamine

Peptides were validated at a 1% False Discovery Rate and manuallyvalidated to ensure high sequence coverage and exclude alternativehypotheses (Aspartic acid 4 Glutamine or CrotonaldehydeCys/His+Carbamidomethylation). All peptides containing a Ser→BMAA whichcould be validated by site specific mass fragmentation are listed belowin Table 6, and the corresponding wild-type sequence fragments (withoutBMAA incorporation) are listed below in Table 7.

TABLE 6 Validated Peptides Containing BMAA Protein (Accession # otherSequence No.) Protein Control ALS mods AYHEQLSVAEITS*S**C*F Q9NY65Human Tubulin alpha- 1 3 1 EPNSQMVK (SEQ ID NO: 24) 8 chain protein(carbami- sequence fragment domethyl) DLYANTVLSGG*S*TMYPG Q562R1Human Beta-actin-like 0 1 0 IADR (SEQ ID NO: 25) protein 2 proteinsequence fragment DTI*C*EE*S*LR (SEQ ID Q8NG31 Human Kinetochore 0 1 1NO: 26) scaffold 1 protein (crotonal- sequence fragment dehyde)ESVSS*S*DR (SEQ ID P40145 Human Adenylate 0 1 0 NO: 27)cyclase type 8 protein sequence fragment GYECILNIQG*S*EQR (SEQ Q9HCM2Human Plexin 4A 1 0 0 ID NO: 28) protein sequence fragment fragmentHTGPGLLSMANSGPNTNG* Q9UNP9 Human Peptidyl- 4 2 0 S*QFFLTCDK (SEQ IDprolyl cis-trans NO: 29) isomerase E peptide sequence fragmentKATYVYET*S*GPNLSDNK A6NE01 Human Protein 1 0 0 SGQK (SEQ ID NO: 30)FAM186A protein sequence fragment KGMW*S*EGNGSHTIR Q7KZF4 Human 0 1 0(SEQ ID NO: 31) Staphylococcal nuclease domain- containing protein 1protein sequence fragment KT*S*TDFSEVIK (SEQ ID Q01484 Human Ankyrin-2 01 0 NO: 32) protein sequence fragment LGQG*S*GQGPK (SEQ ID Q9BSW3Human EF-hand 1 0 0 NO: 33) calcium-binding domain-containingprotein 4B protein sequence fragment LLLGT*S*GEGK (SEQ ID Q9Y4C4Human Malignant 0 1 0 NO: 34) fibrous histiocytoma- amplified sequence 1protein sequence fragment LSLMLDEG*S*SCPTPAK Q9ULV5 Human Heat shock 0 10 (SEQ ID NO: 35) factor protein 4 protein sequence fragmentMSDILRELLCV*S*EK (SEQ P49441 Human Inositol 1 0 0 ID NO: 36)polyphosphate 1- phosphatase protein sequence fragmentNTA*S*HTAAAAR (SEQ ID Q9Y666 Human Solute carrier 0 1 0 NO: 37)family 12 member 7 protein sequence fragment Q*S*ELSAEESPEK (SEQ IDQ567U6 Human Coiled-coil 1 2 0 NO: 38) domain-containingprotein 93 protein sequence fragment *S**C*TAADTAAQITQR Q29960Human HLA class I 2 1 1 (SEQ ID NO: 39) histocompatibility (crotonal-antigen, Cw-16 alpha dehyde) chain protein sequence fragmentSGGGGNFVL*S*TSLVGYL Q9UKG9 Human Peroxisomal 1 0 0 R (SEQ ID NO: 40)carnitine O- octanoyltransferase protein sequence fragment*S*GTSIPSAGK (SEQ ID Q7Z5P9 Human Mucin-19 1 0 0 NO: 41)protein sequence fragment *S*LAGPAGAAPAPGLGAA Q8NES3 Human Beta-1,3-N- 10 0 AAAPGALVR (SEQ ID acetylglucosaminyltra NO: 42)nsferase lunatic fringe protein sequence fragment SLFPPWTFQFQ*S*GDLEEKQ8NDH2 Human coiled-coil 0 1 0 (SEQ ID NO: 43) domain-containingprotein 168 protein sequence fragment SLGGAVGSVA*S*GAR Q8NHG8Human E3 ubiquitin- 1 0 0 (SEQ ID NO: 44) protein ligase ZNRF2protein sequence fragment *S*PVTFLSDLR (SEQ ID A5YKK6 CCR4-NOT 1 0 0NO: 45) transcription complex subunit 1 protein sequence fragment*S*TLVHSLFLTDLYK (SEQ Q99719 Human Septin-5 3 0 0 ID NO: 46)protein sequence fragment VEELIE*S*EAPPK (SEQ ID Q96B23 Human 1 0 0NO: 47) Uncharacterized Protein C18orf25 protein sequence fragmentV*S*ELEDFINGPNNAHIQQ P53675 Human Clathrin heavy 5 3 0VGDR (SEQ ID NO: 48) chain 2 protein sequence fragmentYI*S*DLK (SEQ ID NO: 49) P55774 Human C-C motif 0 1 0chemokine 18 protein sequence fragment *S* = Ser→BMAA *C* =Carbamidomethylation or Crotonaldehyde

TABLE 7 Wild Type Sequences for Peptides Identified in Table 6 ProteinSEQ (Accession Sequence ID NO No.) Protein AYHEQLSVAEITSSCFEPNSQMVKSEQ ID Q9NY65 Human Tubulin alpha-8 NO: 50 chain protein sequencefragment DLYANTVLSGGSTMYPGIADR SEQ ID Q562R1 Human Beta-actin-likeNO: 51 protein 2 protein sequence fragment DTICEESLR SEQ ID Q8NG31Human Kinetochore NO: 52 scaffold 1 protein sequence fragment ESVSSSDRSEQ ID P40145 Human Adenylate cyclase NO: 53 type 8 protein sequencefragment GYECILNIQGSEQR SEQ ID Q9HCM2 Human Plexin 4A protein NO: 54sequence fragment fragment HTGPGLLSMANSGPNTNGSQFFLTC SEQ ID Q9UNP9Human Peptidyl-prolyl cis- DK NO: 55 trans isomerase E peptidesequence fragment KATYVYETSGPNLSDNKSGQK SEQ ID A6NE01Human Protein FAM186A NO: 56 protein sequence fragment KGMWSEGNGSHTIRSEQ ID Q7KZF4 Human Staphylococcal NO: 57 nuclease domain-containing protein 1 protein sequence fragment KTSTDFSEVIK SEQ ID Q01484Human Ankyrin-2 protein NO: 58 sequence fragment LGQGSGQGPK SEQ IDQ9BSW3 Human EF-hand calcium- NO: 59 binding domain-containing protein 4B protein sequence fragment LLLGTSGEGK SEQ ID Q9Y4C4Human Malignant fibrous NO: 60 histiocytoma-amplified sequence 1 proteinsequence fragment LSLMLDEGSSCPTPAK SEQ ID Q9ULV5 Human Heat shock factorNO: 61 protein 4 protein sequence fragment MSDILRELLCVSEK SEQ ID P49441Human Inositol NO: 62 polyphosphate 1- phosphatase proteinsequence fragment NTASHTAAAAR SEQ ID Q9Y666 Human Solute carrier NO: 63family 12 member 7 protein sequence fragment QSELSAEESPEK SEQ ID Q567U6Human Coiled-coil NO: 64 domain-containing protein 93 protein sequencefragment SCTAADTAAQITQR SEQ ID Q29960 Human HLA class I NO: 65histocompatibility antigen, Cw-16 alpha chain protein sequence fragmentSGGGGNFVLSTSLVGYLR SEQ ID Q9UKG9 Human Peroxisomal NO: 66 carnitine o-octanoyltransferase protein sequence fragment SGTSIPSAGK SEQ ID Q7Z5P9Human Mucin-19 protein NO: 67 sequence fragment SLAGPAGAAPAAPGLGAAAAAPGASEQ ID Q8NES3 Human Beta-1,3-N- LVR NO: 68 acetylglucosaminyl-transferase lunatic fringe protein sequence fragment SLFPPWTFQFQSGDLEEKSEQ ID Q8NDH2 Human coiled-coil NO: 69 domain-containing protein168 protein sequence fragment SLGGAVGSVASGAR SEQ ID Q8NHG8Human E3 ubiquitin- NO:70 protein ligase ZNRF2 protein sequence fragmentSPVTFLDSLR SEQ ID A5YKK6 CCR4-NOT transcription NO: 71complex subunit 1 protein sequence fragment STLVHSLFLTDLYK SEQ ID Q99719Human Septin-5 protein NO: 72 sequence fragment VEELIESEAPPK SEQ IDQ96B23 Human Uncharacterized NO: 73 protein C18orf25 proteinsequence fragment VSELEDFINGPNNAHIQQVGDR SEQ ID P53675Human Clathrin heavy NO: 74 chain 2 protein sequence fragment YISDLKSEQ ID P55774 Human C-C motif NO: 75 chemokine 18 proteinsequence fragment

The compositions and methods of the appended claims are not limited inscope by the specific compositions and methods described herein, whichare intended as illustrations of a few aspects of the claims. Anycompositions and methods that are functionally equivalent are intendedto fall within the scope of the claims. Various modifications of thecompositions and methods in addition to those shown and described hereinare intended to fall within the scope of the appended claims. Further,while only certain representative compositions and method stepsdisclosed herein are specifically described, other combinations of thecompositions and method steps also are intended to fall within the scopeof the appended claims, even if not specifically recited. Thus, acombination of steps, elements, components, or constituents may beexplicitly mentioned herein or less, however, other combinations ofsteps, elements, components, and constituents are included, even thoughnot explicitly stated.

The term “comprising” and variations thereof as used herein is usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms. Although the terms “comprising” and“including” have been used herein to describe various embodiments, theterms “consisting essentially of” and “consisting of” can be used inplace of “comprising” and “including” to provide for more specificembodiments of the invention and are also disclosed. Other than wherenoted, all numbers expressing geometries, dimensions, and so forth usedin the specification and claims are to be understood at the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, to be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

1. A polypeptide comprising a sequence that is at least 85% identical toSEQ ID NO: 1, wherein amino acid position 107 comprises a BMAA residuewhich is isotopically labeled and is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least
 100. 2.The polypeptide of claim 1, wherein the ¹³C isotopic enrichment factorfor ^(d)C is at least 80 and the ¹⁵N isotopic enrichment factor for^(f)N is at least
 200. 3. The polypeptide of claim 1, wherein the ¹³Cisotopic enrichment factor for ^(a)C, ^(b)C, and ^(c)C is at least 25.4. The polypeptide of claim 1, wherein the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, and ^(c)C is at least
 80. 5. The polypeptide ofclaim 1, wherein the ¹⁵N isotopic enrichment factor for ^(e)N is atleast
 100. 6. The polypeptide of claim 1, wherein the ¹⁵N isotopicenrichment factor for ^(e)N is at least
 200. 7. The polypeptide of claim1, wherein the sequence is at least 90% identical to SEQ ID NO:
 1. 8.The polypeptide of claim 1, wherein the sequence is at least 95%identical to SEQ ID NO:
 1. 9. The polypeptide of claim 1, wherein thesequence is at least 99% identical to SEQ ID NO:
 1. 10. The polypeptideof claim 1, wherein the sequence is identical to SEQ ID NO:
 1. 11. Apolypeptide comprising a sequence that is at least 85% identical to SEQID NO: 2, wherein amino acid position 107 comprises a BMAA residue whichis isotopically labeled and is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for ^(f)N is at least 100.12. The polypeptide of claim 11, wherein the ¹³C isotopic enrichmentfactor for ^(d)C is at least 80 and the ¹⁵N isotopic enrichment factorfor ^(f)N is at least
 200. 13. The polypeptide of claim 11, wherein the¹³C isotopic enrichment factor for ^(a)C, ^(b)C, and ^(c)C is at least25.
 14. The polypeptide of claim 11, wherein the ¹³C isotopic enrichmentfactor for ^(a)C, ^(b)C, and ^(c)C is at least
 80. 15. The polypeptideof claim 11, wherein the ¹⁵N isotopic enrichment factor for ^(e)N is atleast
 100. 16. The polypeptide of claim 11, wherein the ¹⁵N isotopicenrichment factor for ^(e)N is at least
 200. 17. The polypeptide ofclaim 11, wherein the sequence is at least 90% identical to SEQ ID NO:2.
 18. The polypeptide of claim 11, wherein the sequence is at least 95%identical to SEQ ID NO:
 2. 19. The polypeptide of claim 11, wherein thesequence is at least 99% identical to SEQ ID NO:
 2. 20. The polypeptideof claim 11, wherein the sequence is identical to SEQ ID NO:
 2. 21.-111.(canceled)